Team:HokkaidoU Japan/Notebook/plastic Week 8

Digestion

We digested three samples.
Digestions of PhaC and pSB1C3 with XbaI and SpeI.

Digestion of PhaA with XbaI site and SpeI site.

And digestion of PhaB with XbaI site and SpeI site.

Electrophoresis

digestion result

We confirmed whether PhaC was digested correctly, and phaA and phaB were done PCR correctly by electrophoresis.

Gel extraction

We confirmed succession of digestion by electrophoresis, then DNA were extracted from TBE gel. And we used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation result

The DNA extracted yesterday were condensed by Ethanol precipitation.

Ligation

We ligated phaA with pSB1C3 and phaB with pSB1C3.

Transformation

The ligated DNA were transformed into E.coli (strain: _____). E.Coli solution was spread on LB_.

Colony PCR

We confirmed whether the ligation of yesterday went well by colony PCR.

Colony PCR result

Colony PCR result

Colony PCR

We did colony PCR of PhaA and PhaB again.

Colony PCR result

Colony PCR result

From the result, we thought that we failed to ligate PhaA with pSB1C3 and PhaB with pSB1C3. So we decided to ligated them again.

Ligation

We ligated phaA and phaB with pSB1C3.

Colony PCR

We confirmed the length of PhaA on pSB1C3 and PhaB on pSB1C3 by colony PCR.
The result showed only PhaA and pSB1C3 were ligated correctly.

Colony PCR result

Colony PCR result

Liquid culture

We started to cultivate bacteria holds RBS (B0034).

Digestion

We digested PhaC(BBa_K342001) with XbaI and SpeI. The result shows that the digestion succeeded.

digestion result

Plasmid extraction

The plasmid of RBS (BBa_B0034) were extracted.
And then we got 50ul DNA solution.

plasmid extraction result

Digestion

RBS (BBa_B0034) was digested with SpeI and PstI restriction sites.
And also PhaC (BBa_K342001) was digested with XbaI and PstI.

Liquid culture

We started to cultivate.